The substances referred to as “vitamin D” encompass a group of fat-soluble prohormones, as well as metabolites and analogues thereof. The main forms in which vitamin D occurs in the body are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). The latter is the endogenous form of vitamin D, which humans can form in the skin under the influence of sunlight. The former is an exogenous form of vitamin D, taken up with food. In the US, Vitamin D2 is used as the pharmaceutical vitamin D supplement. Unless indicated otherwise, the term Vitamin D in this disclosure refers to any form or forms of Vitamin D, including Vitamin D metabolites such as 25-hydroxy-Vitamin D or 1,25 dihydroxy Vitamin D.
Whilst vitamin D2 and D3 differ in the molecular structure of their side-chains, they share the same biological activity in being prohormones, metabolized in two steps to, ultimately, 1,25 dihydroxy vitamin D (1,25-(OH)-2-Vitamin D) also referred to as calcitriol, or 1,25 dihydroxy cholecalciferol). The preceding metabolite, 25-hydroxy vitamin D (25-(OH)-Vitamin D) or calcidiol, results from conversion in the liver, and is considered the storage form of vitamin D in the body.
Circulating vitamin D consists mainly of 25-(OH)-Vitamin D3 and 25-(OH)-Vitamin D2. Biologically, 25-(OH)-Vitamin D2 is as effective as 25-(OH)-Vitamin D3. The half-life of 25-(OH)-Vitamin D2 in the circulation is shorter. For clinical practice the use of a 25-(OH)-Vitamin D assay that measures both 25-(OH)-Vitamin D3 as well as 25-(OH)-Vitamin D2 is recommended (1).
Vitamin D has long been recognized as an important substance, the active form of which plays a role in the formation and maintenance of bone, as well as in other processes in the human or animal body. Thus, it serves to increase the flow of calcium into the bloodstream, by promoting absorption of calcium and phosphorus from food in the intestines, and re-absorption of calcium in the kidneys; enabling normal mineralization of bone and preventing hypocalcemic tetany. It is also necessary for bone growth and bone remodeling by osteoblasts and osteoclasts.
Vitamin D deficiency results in impaired bone mineralization and leads to bone softening diseases, rickets in children and osteomalacia in adults, and possibly contributes to osteoporosis.
Vitamin D plays a number of other roles in human health including inhibition of calcitonin release from the thyroid gland. Calcitonin acts directly on osteoclasts, resulting in inhibition of bone resorption and cartilage degradation. Vitamin D can also inhibit parathyroid hormone secretion from the parathyroid gland, modulate neuromuscular and immune function and reduce inflammation. Thus, it is of the essence for a person's or animal's health to have an adequate level of vitamin D.
Yet, excess of vitamin D (which may occur as a result of overdosing) is toxic. Some symptoms of vitamin D toxicity are hypercalcaemia (an elevated level of calcium in the blood) caused by increased intestinal calcium absorption. Vitamin D toxicity is known to be a cause of high blood pressure. Gastrointestinal symptoms of vitamin D toxicity can include anorexia, nausea, and vomiting. These symptoms are often followed by polyuria (excessive production of urine), polydipsia (increased thirst), weakness, nervousness, pruritus (itch), and eventually renal failure.
Clearly, it is important to be able to diagnose subjects for a possible vitamin D deficiency. It is also important, particularly for subjects that are on vitamin D supplementation, to be able to test subjects for a potential excess of vitamin D. The serum level of total 25-(OH)-Vitamin D is considered to be the primary indicator of the vitamin D status (2) However, this notion has been disputed.
Almost all circulating 25-(OH)-Vitamin D in serum is bound by Vitamin D Binding Protein (88%) and Albumin (12%). Vitamin D Binding Protein (DBP) is a major component of serum, with a concentration of 250-400 mg/L of serum. Only a small portion, about 2%, of the Vitamin D binding sites of DBP is occupied. A very small fraction, 0.04% of the 25-(OH)-Vitamin D, circulates in the free, non-protein bound form.
The concentration of DBP is not constant in all people and can be influenced by other factors including pregnancy, the use of oral contraceptives, renal disease and liver disease. Knowledge of the concentration of the DBP is crucial for accurate assessment of the patient's true 25-(OH)-Vitamin D status. For example, a young woman taking oral contraceptives could have a total 25-(OH)-Vitamin D level that was in the normal range. However, due to her elevated DBP, the concentration of Free 25-(OH)-Vitamin D could be markedly depressed, putting her at increased risk for clinical 25-(OH)-Vitamin D insufficiency and all the risks that that condition entails.
It has been shown that the physiological activity of thyroid and steroid hormones in vivo correlates better with their free, non-protein bound fraction, than with the total concentration of the hormone in plasma. Particularly in situations in which the level of binding proteins is elevated or decreased, the measurement of total circulating hormone may lead to a wrong diagnosis. In such situations the measurement of the concentration of the free circulating hormone provides better information. This notion is known as the “free hormone hypothesis”. Mendel (3) suggested that the free hormone hypothesis is “likely to be valid with respect to all tissues for the thyroid hormones, for cortisol, and also for the hydroxylated metabolites of vitamin D.
Bikle et al (4) tested the validity of the free hormone hypothesis for 1,25-(OH)-2-Vitamin D. The data suggested that free 1,25-(OH)-2-Vitamin D levels appeared to be well maintained even in subjects with liver disease and reduced DBP levels, despite a significant decrease of the total 1,25-(OH)-2-Vitamin D.
In a subsequent study on 25-(OH)-Vitamin D the same group recommended to measure free 25-(OH)-Vitamin D in situations with modified concentrations of the binding protein. The author concluded that total vitamin D metabolite measurements may be misleading in the evaluation of the vitamin D status of patients with liver disease, and recommend that free 25-(OH)-Vitamin D levels also be determined before making a diagnosis of vitamin D deficiency. Bikle et al used ultrafiltration to determine the level of free 25-(OH)-Vitamin D (5). This method requires highly purified radiolabeled Vitamin D and tends to overestimate the fraction of free Vitamin D.
Lauridsen et al (6) showed that women with different DBP phenotypes have different concentrations of 1,25(OH)2VitD and 25(OH)VitD. These authors suggest that women with Gc2-2 are Vitamin D sufficient at lower plasma levels of 25(OH)VitD.
Some background art can be referred to regarding the determination of free analytes.
U.S. Pat. No. 4,366,143 describes an invention related to the assay of the free portion of organic substances or ligands that are present in biological fluids in a bound and a free form. The method essentially is a competitive immunoassay wherein, in one step, a labeled ligand, and a specific binder are added to a sample simultaneously. The free portion of the ligand and the labeled ligand compete for reaction with the specific binder, and become bound thereto in proportions which depend on the amount of the free ligand portion present in the sample. A drawback of the disclosed method is that, due to the presence of both the specific binder and the labeled ligand, a plurality of factors is present that are capable of disturbing the equilibrium between bound and free ligand, which makes the method less suitable for use with a free ligand that is present in a relatively low amount as is the case with Vitamin D. In fact, it is not disclosed how to use the assay for the measurement of free Vitamin D.
U.S. Pat. No. 4,292,296 discloses a method for the determination of free analytes in samples containing free analytes and receptor-bound analytes. The method involves two steps, the first being contacting a sample with an absorbent for the analyte to remove analyte from solution. The second step comprises contacting the absorbent-bound analyte with a labeled analyte analogue. Thereupon, the soluble phase is removed from the absorbent, and the amount of label in the bound and washed-away phases are determined. The method is described for determining the concentration of free thyroid hormones.
US patent application 2008/0182341 is related to stabilizing agents that are useful for the measurement of free or unbound analyte concentrations in a fluid. It is suggested that the stabilizers prevent dissociation of the ligand of its binding protein. The reference employs a simultaneous assay procedure, and lists a variety of stabilizing agents. The stabilizing agent is provided not to comprise an alkyl amine fluoro surfactant.
None of the prior art references specifically provides an assay for a determination of Vitamin D that reflects the status of free Vitamin D.
It is noted that assays for Free Vitamin D have been known for decades, but these use methods such as equilibrium dialysis or rate dialysis as their basis. Such methods are acceptable for researchers with highly trained technical staff, but are ill suited for routine laboratories who need high throughput automated tests to reach their financial goals. It is thus desired to provide an assay for free Vitamin D that is capable of being automated and which is suitable for use in point-of-care testing.
The foregoing numbered references are:    1. Hollis B W. Measuring 25-hydroxyvitamin D in a clinical environment: challenges and needs. Am J Clin Nutr. 2008 August; 88(2):507S-510S.    2. Holick M F. Vitamin D: extraskeletal health. Endocrinol Metab Clin North Am. 2010 June; 39(2):381-400.    3 Mendel C M. The free hormone hypothesis: a physiologically based mathematical model. Endocr Rev. 1989 August; 10(3):232-74.    4. Bikle D, Gee E, Halloran B, Haddad J. Free 1,25-Dihydroxyvitamin D Levels in Serum from Normal Subjects, Pregnant Subjects, and Subjects with Liver Disease. J Clin Invest. 1984; 74: 1966-1971.    5. Bikle D, Gee E, Halloran B, Kowalski M A, Ryzen E, Haddad J. Assessment of the free fraction of 25-hydroxyvitamin D in serum and its regulation by albumin and the vitamin D-binding protein. J Clin Endocrinol Metab. 1986 October; 63(4):954-9.    6. Lauridsen A L, Vestergaard P, Hermann A P, Brot C, Heickendorff L, Mosekilde L, Nexo E. Plasma concentrations of 25-hydroxy-vitamin D and 1,25-dihydroxy-vitamin D are related to the phenotype of Gc (vitamin D-binding protein): a cross-sectional study on 595 early postmenopausal women. Calcif Tissue Int. 2005 July; 77(1):15-22.    7. van Hoof H J, Swinkels L M, Ross H A, Sweep C G, Benraad T J. Determination of non-protein-bound plasma 1,25-dihydroxyvitamin D by symmetric (rate) dialysis. Anal Biochem 1998 May 1; 258(2):176-83.